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CN108948211A - 一种基于gd2的嵌合抗原受体及其应用 - Google Patents

一种基于gd2的嵌合抗原受体及其应用 Download PDF

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CN108948211A
CN108948211A CN201810821559.9A CN201810821559A CN108948211A CN 108948211 A CN108948211 A CN 108948211A CN 201810821559 A CN201810821559 A CN 201810821559A CN 108948211 A CN108948211 A CN 108948211A
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CN108948211B (zh
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李昱琛
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Beijing American Kinge Biotech Co Ltd
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Abstract

本发明涉及一种基于GD2嵌合抗原受体，其特征在于，所述嵌合抗原受体包括抗原结合结构域、跨膜结构域、共刺激信号传导结构域、CD3ζ信号传导结构域和自毁结构域串联而成；其中，所述抗原结合结构域结合肿瘤表面抗原，所述肿瘤表面抗原为GD2，所述抗原结合结构域为针对肿瘤表面抗原GD2的单链抗体，所述自毁结构域为胱天蛋白酶9结构域。本发明的嵌合抗原受体实际应用于表达肿瘤特异靶点GD2的IV期神经母细胞瘤病人中，有更小的临床副作用与更高的安全性，可有效缩小实体瘤灶，并于病人体内长期监测到GD2‑CART的存在，有效延长病人整体生存率。

Description

一种基于GD2的嵌合抗原受体及其应用
技术领域
本发明涉及肿瘤的细胞免疫治疗领域，尤其涉及一种基于GD2的嵌合抗原受体及其应用，具体为以肿瘤特异靶点GD2为基础的嵌合抗原受体T(CAR-T)细胞技术的构建方法及其在抗肿瘤治疗中的应用。
背景技术
随着肿瘤免疫学理论和临床技术的发展，嵌合抗原受体T细胞疗法(Chimericantigen receptor T-cell immunotherapy，CAR-T)成为目前最有发展前景的肿瘤免疫疗法之一。一般，嵌合抗原受体CAR由一个肿瘤相关抗原结合区、胞外铰链区、跨膜区域以及胞内信号转导区组成。通常，CAR包含抗体的单链片段可变(Single chain fragmentvariable，scFv)区或对肿瘤相关抗原(tumor associated antigen,TAA)具有特异性的结合结构域，其通过铰链和跨膜区与T细胞信号传导分子的胞质结构域偶联。最常见的淋巴细胞活化部分包括与T细胞效应物功能触发(例如CD3ζ)部分串联的T细胞共刺激结构域。CAR介导的过继性免疫疗法允许CAR-移植的T细胞以非HLA限制性方式直接识别靶肿瘤细胞上的TAA。
神经母细胞瘤是儿童最常见的颅外实体恶性肿瘤，其中有50％的病童被发先有肿瘤大规模的扩散与转移，常规的手术、化疗、放疗与自体干细胞移植对于这群病人的治疗效果有限，即使在控制病情达到缓解后也有80％以上的病人会于一年内复发并且死亡。治疗这些患者的一种方法是通过CAR的表达，对T细胞进行遗传修饰以靶向在肿瘤细胞上表达的抗原。CAR是经设计以人白细胞抗原(human leukocyte antigen,HLA)非依赖性方式识别细胞表面抗原的抗原受体。尝试使用表达CAR的遗传修饰细胞来治疗这些类型的患者已经取得了有前景的成功。
GD2广泛地表现在神经母细胞瘤、黑色素瘤等肿瘤当中，在正常组织中低量且局限性地表达，是一免疫治疗理想的肿瘤抗原，目前在神经母细胞瘤的免疫治疗中，发展较为成熟的有针对GD2的抗体治疗，此一方式在临床上取得初步的成功，但抗体治疗的问题在于，抗体施打后存在于外围血当中较难精准地进入肿瘤组织或肿瘤微小残留的部位，且抗体施打后无法长期存在体内，而此抗GD2抗体为人源-鼠源嵌合的抗体结构，势必会使人体对其产生抗性，增加了再治疗的困难性。
CN 106536563 A公开了包含双唾液酸神经节苷脂(GD2)结合域的嵌合抗原受体(CAR)，所述GD2结合域包含a)具有互补决定区(CDR)的重链可变区；b)具有CDR的轻链可变区；及表达此类CAR的T细胞在一些癌症的治疗中的作用；该专利中的嵌合抗原受体与GD2的结合效果有待提高，其免疫效果也可以进一步提高。
因此，制备一种完全人源化的嵌合抗原受体GD2-CART细胞，除了俱有抗体治疗的优点，且因为T细胞本身的特性，可精准地进入肿瘤组织，并长期的存在于体内，必能提供复发难治的神经母细胞瘤一个更有效的治疗选择。
发明内容
针对目前CAR-T技术治疗肿瘤中靶向不十分理想，以及肿瘤微环境影响CAR-T技术治疗效果的情况，本发明提供一种基于GD2的嵌合抗原受体及其应用，本发明制备的嵌合抗原受体通过将GD2-CAR-T进行基因改造，从而提高了靶点的免疫长效性与安全性，增强了CAR-T细胞的治疗效果。
为达此目的，本发明采用以下技术方案：
一方面，本发明提供一种基于GD2的嵌合抗原受体，所述嵌合抗原受体包括抗原结合结构域、跨膜结构域、共刺激信号传导结构域、CD3ζ信号传导结构域和自毁结构域串联而成；
其中，所述抗原结合结构域结合肿瘤表面抗原，所述肿瘤表面抗原为GD2，所述抗原结合结构域为针对肿瘤表面抗原GD2的单链抗体，所述肿瘤表面抗原GD2的单链抗体的氨基酸序列选自：
(a)如SEQ ID NO.1-3所示的氨基酸序列；或者，
(b)如SEQ ID NO.1-3所示的氨基酸序列经过一个或多个氨基酸的取代、添加或缺失而形成的，能够特异结合在嵌合抗原受体上，具有结合GD2和诱导T细胞信号传导功能的氨基酸序列。
本发明中，通过对抗原结合结构域的单链抗体与CAR的基因结构进行特定的改造，从而使得基因修改过后的CAR-T细胞能够特异的结合在肿瘤的GD抗原上，获得相对比较适度的信号刺激，发挥有效的杀伤作用，同时缓慢释放免疫因子，降低免疫因子风暴危险，相比于其他嵌合抗原受体和其他肿瘤抗原有更好的效果与安全性。
所述针对肿瘤表面抗原GD2的单链抗体氨基酸序列(SEQ ID NO.1-3)如下：
SEQ ID NO.1所示的氨基酸序列：
HPAFLLIPQVQLVESGPGVVQPGRSLRISCAVSGFSVTNYGVHWVRQPPGKGLEWLGVIWAGGITNYNSAFMSRLTISKDNSKNTVYLQMNSLRAEDTAMYYCASRGGHYGYALDYWGQGTLVTVSSGSTSGSGKPGSSEGSTKGEIVMTQTPATLSVSAGERVTITCKASQSVSNDVTWYQQKPGQAPRLLIYSASNRYSGVPARFSGSGYGTEFTFTISSVQSEDFAVYFCQQDYSSFGQGTKLEIK；
SEQ ID NO.2所示的氨基酸序列：
EVQLVQSGAEVEKPGASVKISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGGTSYNQKFKGRATLTVDKSTSTAYMHLKSLRSEDTAVYYCVSGMEYWGQGTSVTVSSGSTSGSGKPGSSEGSTKGDVVMTQTPLSLPVTPGEPASISCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELK；
SEQ ID NO.3所示的氨基酸序列：
AFLLIPEVKLVESGGGLVLPGDSLRLSCATSEFTFTDYYMTWVRQPPRKALEWLGFIRNRANGYTTEYNPSVKGRFTISRDNSQSILYLQMNTLRTEDSATYYCARVSNWAFDYWGQGTTLTVSSGSTSGSGKPGSSEGSTKGDVVMTQTPLSLPVSLGDQASISCRSSQSLLKNNGNTFLHWYLQKSGQSPKLLIYKVSNRLSGVPDRFSGSGSGTYFTLKISRVEAEDLGVYFCSQSTHIPYTFGGGTKLEIK.
本发明中，所述针对肿瘤表面抗原GD2的CAR信号结构进行了特异性改造，也能快速与不同的GD2单链抗体进行改造，使得改造后的CAR表达出来的免疫刺激力更强。
根据本发明，所述如SEQ ID NO.1-3所示的氨基酸序列经过一个或多个氨基酸的取代、添加或缺失而形成的氨基酸序列与SEQ ID NO.1-3所示的氨基酸序列具有至少90％同一性，优选为95％同一性的氨基酸序列，所述改造后的氨基酸序列仍然能够特异结合在嵌合抗原受体上，具有结合GD2和诱导T细胞信号传导功能。
所述90％同一性例如可以是90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％。
根据本发明，所述跨膜结构域为CD28跨膜结构域(CD28细胞膜外和CD28细胞膜传导结构域)和/或CD8α跨膜结构域，在一些具体实施方案中，可以通过氨基酸替换来选择或修饰跨膜结构域。
根据本发明，所述共刺激信号传导结构域为CD28信号传导结构域和4-1BB细胞内信号传导结构域的组合，所述CD28细胞外、细胞膜和细胞内传导结构域组合成CD28全信号传导结构域，所述CD28全信号传导结构域和4-1BB信号传导结构域的排列，本领域技术人员可以根据需要进行调整，CD28全信号传导结构域和4-1BB信号传导结构域不同的排列不会对所述嵌合抗原受体产生影响，本申请采用CD28-4-1BB的顺序组合所述共刺激信号传导结构域的氨基酸序列如SEQ ID NO.4所示，具体序列如下：
IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSASGGGGSGGGGSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.
其中的linker序列为多个GGGGS的重复序列。
根据本发明，所述CD28全信号传导结构域包括CD28细胞外信号、CD28细胞膜信号和CD28细胞内信号；所述CD28细胞外信号序列如SEQ ID NO.5所示，具体如下：IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP；所述CD28细胞膜信号序列如SEQ ID NO.6所示，具体如下：FWVLVVVGGVLACYSLLVTVAFIIFWV；所述CD28细胞内信号序列如SEQ ID NO.7所示，具体如下：RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSAS；
根据本发明，所述4-1BB细胞内信号序列如SEQ ID NO.8所示，具体如下：VVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.
根据本发明，所述自毁结构域为包含胱天蛋白酶9结构域，所述胱天蛋白酶9结构域的氨基酸序列如SEQ ID NO.9所示，所述胱天蛋白酶9结构域的氨基酸序列(SEQ IDNO.9)如下：
GSGATNFSLLKQAGDVEENPGPMGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGGSGGGGSGAMVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELARQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSAS.
根据本发明，所述自毁结构域通过2A序列与CD3ζ信号传导结构域相串联，所述2A序列会使所述自毁结构域表达的蛋白与所述嵌合抗原受体蛋白断裂开，从而使得所述嵌合抗原受体能够发挥作用，而通过注入激活剂，从而使得自毁结构域激活，从而导致嵌合抗原受体失去作用。
根据本发明，所述嵌合抗原受体还包括信号肽，所述信号肽为能够指导嵌合抗原受体跨膜转移的信号肽都是可行的，本领域技术人员可以根据需要选择本领域常规的信号肽，所述信号肽为Secretory信号肽，所述Secretory信号肽的氨基酸序列如SEQ ID NO.10-11所示，所述Secretory信号肽的氨基酸序列(SEQ ID NO.10)如下：MLLLVTSLLLCELPHPAFLLIP；或所述Secretory信号肽的氨基酸序列(SEQ ID NO.11)如下：MALPVTALLLPLALLLHAARP.
本发明的嵌合抗原受体还可以包括铰链区，所述铰链区本领域技术人员可以根据实际情况进行选择，在此不做特殊限定，铰链区的存在不会对本发明的嵌合抗原受体的性能产生影响。
本发明的嵌合抗原受体还包括启动子，所述启动子可以为EFla或任何一种高表达的启动子，本领域技术人员可以根据实际情况进行选择，在此不做特殊限定，启动子的存在不会对本发明的嵌合抗原受体的性能产生影响。
根据本发明，所述嵌合抗原受体包括信号肽、抗原结合结构域、跨膜结构域、共刺激信号传导结构域、CD3ζ信号传导结构域、2A序列和自毁结构域串联而成。
作为优选技术方案，所述嵌合抗原受体为Secretory信号肽，GD2抗原结合结构域，CD8α和/或CD28跨膜结构域，CD28全信号传导结构域、4-1BB细胞内信号传导结构域，CD3ζ信号传导结构域、2A序列和胱天蛋白酶9结构域串联而成，具体排列如下：
Secretory-GD2scFv-CD28-4-1BB-CD3ζ-2A-FBKP.Casp9。
根据本发明，所述嵌合抗原受体Secretory-GD2scFv-CD28-4-1BB-CD3ζ-2A-FBKP.Casp9的氨基酸序列如SEQ ID NO.12-14所示，具体如下：
SEQ ID NO.12所示的氨基酸序列：
MLLLVTSLLLCELPHPAFLLIPQVQLVESGPGVVQPGRSLRISCAVSGFSVTNYGVHWVRQPPGKGLEWLGVIWAGGITNYNSAFMSRLTISKDNSKNTVYLQMNSLRAEDTAMYYCASRGGHYGYALDYWGQGTLVTVSSGSTSGSGKPGSSEGSTKGEIVMTQTPATLSVSAGERVTITCKASQSVSNDVTWYQQKPGQAPRLLIYSASNRYSGVPARFSGSGYGTEFTFTISSVQSEDFAVYFCQQDYSSFGQGTKLEIKAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSASGGGGSGGGGSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGGGGSGGGGSGGGGSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRTSGSGATNFSLLKQAGDVEENPGPMGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGGSGGGGSGAMVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELARQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSAS；
SEQ ID NO.13所示的氨基酸序列：
MLLLVTSLLLCELPEVQLVQSGAEVEKPGASVKISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGGTSYNQKFKGRATLTVDKSTSTAYMHLKSLRSEDTAVYYCVSGMEYWGQGTSVTVSSGSTSGSGKPGSSEGSTKGDVVMTQTPLSLPVTPGEPASISCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELKAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSASGGGGSGGGGSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGGGGSGGGGSGGGGSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRTSGSGATNFSLLKQAGDVEENPGPMGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGGSGGGGSGAMVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELARQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSAS；
SEQ ID NO.14所示的氨基酸序列：
MLLLVTSLLLCELPAFLLIPEVKLVESGGGLVLPGDSLRLSCATSEFTFTDYYMTWVRQPPRKALEWLGFIRNRANGYTTEYNPSVKGRFTISRDNSQSILYLQMNTLRTEDSATYYCARVSNWAFDYWGQGTTLTVSSGSTSGSGKPGSSEGSTKGDVVMTQTPLSLPVSLGDQASISCRSSQSLLKNNGNTFLHWYLQKSGQSPKLLIYKVSNRLSGVPDRFSGSGSGTYFTLKISRVEAEDLGVYFCSQSTHIPYTFGGGTKLEIKAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSASGGGGSGGGGSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGGGGSGGGGSGGGGSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRTSGSGATNFSLLKQAGDVEENPGPMGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGGSGGGGSGAMVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELARQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSAS.
第二方面，本发明提供一种病毒载体，包括第一方面所述的嵌合抗原受体。
根据本发明，所述病毒载体为慢病毒载体和/或逆转录病毒载体，优选为慢病毒载体。
第三方面，本发明提供一种T细胞，采用如第一方面所述的嵌合抗原受体通过其编码的核酸序列转染到T细胞中表达。
根据本发明，所述转染的方式为通过病毒载体和/或真核表达质粒转染到T细胞，优选为通过病毒载体转染到T细胞。
本发明中，所述T细胞具有良好的靶向杀伤作用，同时能够释放低剂量免疫因子，具备低毒性高免疫杀伤反应性质。
第四方面，本发明提供一种重组慢病毒，将包含如第二方面所述的病毒载体与包装辅助质粒pNHP和pHEF-VSVG共转染哺乳细胞得到的重组慢病毒。
根据本发明，所述哺乳细胞为293细胞、293T细胞或TE671细胞中的任意一种或至少两种的组合。
第五方面，本发明提供一种组合物，所述组合物包括如第一方面所述的嵌合抗原受体和/或如第四方面所述的重组慢病毒。
第六方面，本发明提供如第一方面所述的嵌合抗原受体、如第二方面所述的病毒载体、如第三方面所述的T细胞、如第四方面所述的重组慢病毒或如第五方面所述的组合物在制备嵌合抗原受体T细胞及其在肿瘤治疗药物中的应用；
优选地，所述肿瘤为GD2特异抗原表达的肿瘤疾病，所述GD2特异抗原表达的肿瘤疾病为神经母细胞瘤。
与现有技术相比，本发明具有如下有益效果：
(1)本发明的嵌合抗原受体通过对所述针对肿瘤表面抗原GD2的嵌合抗原受体的T细胞内共刺激信号传导结构域进行特定的基因改造，改造后的嵌合抗原受体在专一性的与GD2结合后有更好的反应效果，使得CAR-T细胞对肿瘤产生更强的免疫反应；
(2)本发明实际应用与人体后，相较于其他GD2的嵌合抗原受体T细胞，具有更高的安全性，即使发生了不良反应，由于具有诱导凋亡机制信号，也能透过诱导CAR-T细胞凋亡的药物将其撤除；
(3)本发明的嵌合抗原受体在进行CAR-T细胞回输后，可于体内长期监测到CAR-T的存在，证明其具有长效性，能使病人达到长期缓解的效用。
附图说明
图1为本发明的嵌合抗原受体的合成基因序列图谱；
图2为安全有效的慢病毒载体靶向CART应用，其中Type1为实施例1制备的嵌合抗原受体，Type2为实施例2制备的嵌合抗原受体，Type3为实施例3制备的嵌合抗原受体；
图3为三种GD2ScFv CART体外杀伤结果图，其中Type1为实施例1制备的嵌合抗原受体，Type2为实施例2制备的嵌合抗原受体，Type3为实施例3制备的嵌合抗原受体；
图4为神经母细胞瘤病人肿瘤切片免疫组化染色结果图，其中，图4(a)为肿瘤切片阴性对照，图4(b)为GD2阳性表达；
图5为GD2-CART回输神经母细胞瘤病人后毒性反应结果图；
图6为GD2-CART回输后体内CAR拷贝数侦测曲线图；
图7为神经母细胞瘤病人GD2-CART回输后以B超检测肿瘤体积变化曲线图；
图8为神经母细胞瘤病人GD2-CART回输前后腹腔肿块增强CT图片，图8(a)为回输前，图8(b)为回输后。
具体实施方式
为更进一步阐述本发明所采取的技术手段及其效果，以下结合附图并通过具体实施方式来进一步说明本发明的技术方案，但本发明并非局限在实施例范围内。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。
实施例1：嵌合抗原受体的构建(I)
(1)通过全基因合成Secretory信号肽、GD2抗原结合结构域，CD28细胞外以及跨膜结构域，CD28细胞内信号传导结构域和4-1BB信号传导结构域，CD3ζ信号传导结构域、2A序列和胱天蛋白酶9结构域，如图1所示，即Secretory-GD2scFv-CD28-4-1BB-CD3ζ-2A-FBKP.Casp9；
所述嵌合抗原受体的氨基酸序列SEQ ID NO.12如下：
MLLLVTSLLLCELPHPAFLLIPQVQLVESGPGVVQPGRSLRISCAVSGFSVTNYGVHWVRQPPGKGLEWLGVIWAGGITNYNSAFMSRLTISKDNSKNTVYLQMNSLRAEDTAMYYCASRGGHYGYALDYWGQGTLVTVSSGSTSGSGKPGSSEGSTKGEIVMTQTPATLSVSAGERVTITCKASQSVSNDVTWYQQKPGQAPRLLIYSASNRYSGVPARFSGSGYGTEFTFTISSVQSEDFAVYFCQQDYSSFGQGTKLEIKAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSASGGGGSGGGGSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGGGGSGGGGSGGGGSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRTSGSGATNFSLLKQAGDVEENPGPMGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGGSGGGGSGAMVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELARQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSAS。
实施例2：嵌合抗原受体的构建(II)
(1)通过全基因合成Secretory信号肽、GD2抗原结合结构域，CD28细胞外和跨膜结构域，CD28信号传导结构域和4-1BB信号传导结构域，CD3ζ信号传导结构域、2A序列和胱天蛋白酶9结构域，即Secretory-GD2scFv-CD28-4-1BB-CD3ζ-2A-FBKP.Casp9；
所述嵌合抗原受体的氨基酸序列SEQ ID NO.13如下：
MLLLVTSLLLCELPEVQLVQSGAEVEKPGASVKISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGGTSYNQKFKGRATLTVDKSTSTAYMHLKSLRSEDTAVYYCVSGMEYWGQGTSVTVSSGSTSGSGKPGSSEGSTKGDVVMTQTPLSLPVTPGEPASISCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELKAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSASGGGGSGGGGSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGGGGSGGGGSGGGGSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRTSGSGATNFSLLKQAGDVEENPGPMGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGGSGGGGSGAMVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELARQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSAS。
实施例3：嵌合抗原受体的构建(III)
(1)通过全基因合成Secretory信号肽、GD2抗原结合结构域，CD28细胞外和跨膜结构域，CD28信号传导结构域和4-1BB信号传导结构域，CD3ζ信号传导结构域、2A序列和胱天蛋白酶9结构域，即Secretory-GD2scFv-CD28-4-1BB-CD3ζ-2A-FBKP.Casp9；
所述嵌合抗原受体的氨基酸序列SEQ ID NO.14如下：
MLLLVTSLLLCELPAFLLIPEVKLVESGGGLVLPGDSLRLSCATSEFTFTDYYMTWVRQPPRKALEWLGFIRNRANGYTTEYNPSVKGRFTISRDNSQSILYLQMNTLRTEDSATYYCARVSNWAFDYWGQGTTLTVSSGSTSGSGKPGSSEGSTKGDVVMTQTPLSLPVSLGDQASISCRSSQSLLKNNGNTFLHWYLQKSGQSPKLLIYKVSNRLSGVPDRFSGSGSGTYFTLKISRVEAEDLGVYFCSQSTHIPYTFGGGTKLEIKAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSASGGGGSGGGGSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGGGGSGGGGSGGGGSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRTSGSGATNFSLLKQAGDVEENPGPMGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGGSGGGGSGAMVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLELARQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSAS。
实施例4：慢病毒包装
(1)用六孔板分别培养293T细胞，1×106个细胞/孔，培养17-18小时；
(2)加入600μL/孔的新鲜的DMEM，内含10％的FBS；
(3)在无菌离心管中加入以下试剂：每孔取75μL的DMEM加入helper DNA mix(pNHP，pHEF-VSV-G，pHEF-eGFP)以及pTYF CAR DNA载体(实施例1-3)，漩涡振荡；
(4)从每孔板中央吸取7μL的Superfect加至离心管中吹打5次，室温静置7-10分钟；
(5)将离心管中的DNA-Superfect混合液逐滴加入至每个培养孔中，漩涡打匀；
(6)37℃3％CO2培养箱里培养4-5小时；
(7)吸走培养基的培养液，用1.5mL AIM-V冲洗培养基，并加入1.5mL的AIM-V继续培养；
(8)将培养基放回3％CO2培养箱中培养过夜，第二天早上用荧光显微镜观察转染效率。
实施例5：慢病毒的纯化和浓缩
1)病毒纯化
通过离心(1000g，5分钟)除去细胞碎片，得到病毒上清液，用一个0.45微米的低蛋白结合过滤器将病毒上清液过滤，病毒被分装成小份，储存在-80℃；
通常情况下，在每毫升的培养基中，转染细胞可以产生106到107转导单位滴定的慢病毒载体。
2)用过滤器浓缩慢病毒载体
(1)在生物安全柜中，取过滤管，用70％酒精消毒，然后用无菌PBS清洗；
(2)每个过滤管中加入18ml的病毒上清液，然后在2500g下离心30分钟或者直到病毒体积减小20-50倍；
(3)震荡过滤管，然后在400g下，离心2分钟，收集浓缩的病毒到收集杯中，最后将所有管中的病毒集中到一个离心管中。
实施例6：CAR-T细胞的转染
将活化后的T细胞接种于培养液中并且添加10μg/mL的polybrene，培养液含有细胞培养因子IL-2、IL-7与IL-15的AIM-V，加入的浓缩CAR基因慢病毒，以100g离心力的速度，室温离心100分钟后，置于37℃培养24h，加入培养液，培养4天后，将细胞收获并计数，培养2天后输至病人。
其治疗肿瘤的效果如图2所示，能够有效使肿瘤缩小，且安全，具体将通过体外试验和体内试验分别验证。
实施例7：CAR-T细胞的体外杀伤试验
(1)将GD2阳性的肿瘤细胞株以慢病毒载体送入绿色荧光蛋白使其稳定表达；
(2)将非专一性的T细胞或不同于GD2ScFv的CAR-T细胞，与上述肿瘤置于37度5％CO2培养箱共培养24-72h；
(2)以荧光显微镜观察肿瘤细胞存活的情形，死亡的肿瘤细胞没有绿色荧光蛋白的表达，以此评估不同GD2-CAR-T细胞的体外杀伤效率，结果如图3所示；
从图3可以看出，相较于对照组T细胞，三种不同ScFv GD2-CAR-T都有明显的杀伤，又以第三种scFv的效果为最佳，因此证实此发明载体材料可以快速筛选出有效CAR结构，并进行后续临床应用。
实施例8：CAR-T细胞的治疗效果
(1)神经母细胞瘤病人的肿瘤白片由免疫组化染色确认GD2的阳性表达，结果如图4所示，从图4(a)和图4(b)可以区分出GD2表达高的肿瘤染色。
(2)采集病人白血球浓厚液，以Ficoll进行密度梯度离心法分离白血球浓厚液中的外围单个核淋巴细胞，并使用CD3磁珠筛选出T细胞并加入抗CD28的抗体进行T细胞活化，以1×106CART细胞数/公斤体重进行后续GD2-CART制备；
(3)回输前病人以小剂量化疗进行预处理，预处理方案为环磷酰胺250mg/m2使用三天，氟达拉滨25mg/m2使用三天，预处理与CART输注间隔24h，总体3天(化疗方案可以依据病人情况修改，此实施例仅仅作为一个列举)；
(4)以静脉注射方式回输CART细胞；
(5)回输后由临床医生对病人进行监测与毒性反应评估，结果如图5所示；
从图5统计可看出GD2-CART的安全性，回输后有24％的病人无任何不良反应，50％的病人有一级不良反应，26％病人有二级不良反应，不良反应包括了发烧、疲倦、皮疹与低血压等等，临床上都可以有效控制；
(6)回输后定期抽取病人少量外围血，分离外围单个核淋巴细胞后抽取细胞染色体DNA(gDNA)，使用专一性引物以qPCR方式定量外围血中CAR拷贝数，结果如图6所示；
从图6可看出病人回输后20天左右体内CAR值达到高峰，并可于体内维持约半年；
(7)GD2-CART回输后以影像学的方式对肿瘤大小进行评估，结果如图7和图8所示；
从图7可以看出，以B超对肿瘤进行监测，此病人在回输GD2-CART后28天腹腔与纵隔肿块相较回输前缩小约95％，并持续维持半年，此病人至今依然状况稳定；而从图8(a)-图8(b)可看出，另一病人在回输后两个月以增强CT扫描腹部肿块，肿块图8(a)原来为长1.9cm，宽4.9cm缩小至图8(b)长1.9cm，宽3.2cm。
综上所述，本发明的GD2-CART相比于其他嵌合抗原受体和其他肿瘤抗原有更好的效果，且具有安全性和长效性，对于复发难治的四期神经母细胞瘤病人确实达到很好的疗效。
申请人声明，本发明通过上述实施例来说明本发明的详细方法，但本发明并不局限于上述详细方法，即不意味着本发明必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。
SEQUENCE LISTING
<110> 北京美康基免生物科技有限公司
<120> 一种基于GD2的嵌合抗原受体及其应用
<130> 2018
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Gly Thr Asp Gly Cys Pro Val Ser Val Glu Lys Ile Val Asn Ile Phe
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Asn Gly Thr Ser Cys Pro Ser Leu Gly Gly Lys Pro Lys Leu Phe Phe
275 280 285
Ile Gln Ala Cys Gly Gly Glu Gln Lys Asp His Gly Phe Glu Val Ala
290 295 300
Ser Thr Ser Pro Glu Asp Glu Ser Pro Gly Ser Asn Pro Glu Pro Asp
305 310 315 320
Ala Thr Pro Phe Gln Glu Gly Leu Arg Thr Phe Asp Gln Leu Asp Ala
325 330 335
Ile Ser Ser Leu Pro Thr Pro Ser Asp Ile Phe Val Ser Tyr Ser Thr
340 345 350
Phe Pro Gly Phe Val Ser Trp Arg Asp Pro Lys Ser Gly Ser Trp Tyr
355 360 365
Val Glu Thr Leu Asp Asp Ile Phe Glu Gln Trp Ala His Ser Glu Asp
370 375 380
Leu Gln Ser Leu Leu Leu Arg Val Ala Asn Ala Val Ser Val Lys Gly
385 390 395 400
Ile Tyr Lys Gln Met Pro Gly Cys Phe Asn Phe Leu Arg Lys Lys Leu
405 410 415
Phe Phe Lys Thr Ser Ala Ser
420
<210> 10
<211> 22
<212> PRT
<213> 人工合成
<400> 10
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro
20
<210> 11
<211> 21
<212> PRT
<213> 人工合成
<400> 11
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro
20
<210> 12
<211> 981
<212> PRT
<213> 人工合成
<400> 12
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro Gln Val Gln Leu Val Glu Ser Gly Pro Gly
20 25 30
Val Val Gln Pro Gly Arg Ser Leu Arg Ile Ser Cys Ala Val Ser Gly
35 40 45
Phe Ser Val Thr Asn Tyr Gly Val His Trp Val Arg Gln Pro Pro Gly
50 55 60
Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Ala Gly Gly Ile Thr Asn
65 70 75 80
Tyr Asn Ser Ala Phe Met Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser
85 90 95
Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
100 105 110
Ala Met Tyr Tyr Cys Ala Ser Arg Gly Gly His Tyr Gly Tyr Ala Leu
115 120 125
Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Ser Thr
130 135 140
Ser Gly Ser Gly Lys Pro Gly Ser Ser Glu Gly Ser Thr Lys Gly Glu
145 150 155 160
Ile Val Met Thr Gln Thr Pro Ala Thr Leu Ser Val Ser Ala Gly Glu
165 170 175
Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Asp Val
180 185 190
Thr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr
195 200 205
Ser Ala Ser Asn Arg Tyr Ser Gly Val Pro Ala Arg Phe Ser Gly Ser
210 215 220
Gly Tyr Gly Thr Glu Phe Thr Phe Thr Ile Ser Ser Val Gln Ser Glu
225 230 235 240
Asp Phe Ala Val Tyr Phe Cys Gln Gln Asp Tyr Ser Ser Phe Gly Gln
245 250 255
Gly Thr Lys Leu Glu Ile Lys Ala Ala Ala Ile Glu Val Met Tyr Pro
260 265 270
Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly Thr Ile Ile His Val
275 280 285
Lys Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser Lys
290 295 300
Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser
305 310 315 320
Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg
325 330 335
Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro
340 345 350
Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe
355 360 365
Ala Ala Tyr Arg Ser Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
370 375 380
Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
385 390 395 400
Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
405 410 415
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Gly Gly Gly
420 425 430
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Val Lys Phe
435 440 445
Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu
450 455 460
Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp
465 470 475 480
Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys
485 490 495
Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala
500 505 510
Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys
515 520 525
Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr
530 535 540
Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Thr Ser Gly Ser
545 550 555 560
Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu
565 570 575
Asn Pro Gly Pro Met Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp
580 585 590
Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr
595 600 605
Gly Met Leu Glu Asp Gly Lys Lys Val Asp Ser Ser Arg Asp Arg Asn
610 615 620
Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp
625 630 635 640
Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr
645 650 655
Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile
660 665 670
Pro Pro His Ala Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu
675 680 685
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ala Met Val Gly Ala
690 695 700
Leu Glu Ser Leu Arg Gly Asn Ala Asp Leu Ala Tyr Ile Leu Ser Met
705 710 715 720
Glu Pro Cys Gly His Cys Leu Ile Ile Asn Asn Val Asn Phe Cys Arg
725 730 735
Glu Ser Gly Leu Arg Thr Arg Thr Gly Ser Asn Ile Asp Cys Glu Lys
740 745 750
Leu Arg Arg Arg Phe Ser Ser Leu His Phe Met Val Glu Val Lys Gly
755 760 765
Asp Leu Thr Ala Lys Lys Met Val Leu Ala Leu Leu Glu Leu Ala Arg
770 775 780
Gln Asp His Gly Ala Leu Asp Cys Cys Val Val Val Ile Leu Ser His
785 790 795 800
Gly Cys Gln Ala Ser His Leu Gln Phe Pro Gly Ala Val Tyr Gly Thr
805 810 815
Asp Gly Cys Pro Val Ser Val Glu Lys Ile Val Asn Ile Phe Asn Gly
820 825 830
Thr Ser Cys Pro Ser Leu Gly Gly Lys Pro Lys Leu Phe Phe Ile Gln
835 840 845
Ala Cys Gly Gly Glu Gln Lys Asp His Gly Phe Glu Val Ala Ser Thr
850 855 860
Ser Pro Glu Asp Glu Ser Pro Gly Ser Asn Pro Glu Pro Asp Ala Thr
865 870 875 880
Pro Phe Gln Glu Gly Leu Arg Thr Phe Asp Gln Leu Asp Ala Ile Ser
885 890 895
Ser Leu Pro Thr Pro Ser Asp Ile Phe Val Ser Tyr Ser Thr Phe Pro
900 905 910
Gly Phe Val Ser Trp Arg Asp Pro Lys Ser Gly Ser Trp Tyr Val Glu
915 920 925
Thr Leu Asp Asp Ile Phe Glu Gln Trp Ala His Ser Glu Asp Leu Gln
930 935 940
Ser Leu Leu Leu Arg Val Ala Asn Ala Val Ser Val Lys Gly Ile Tyr
945 950 955 960
Lys Gln Met Pro Gly Cys Phe Asn Phe Leu Arg Lys Lys Leu Phe Phe
965 970 975
Lys Thr Ser Ala Ser
980
<210> 13
<211> 976
<212> PRT
<213> 人工合成
<400> 13
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro Glu Val
1 5 10 15
Gln Leu Val Gln Ser Gly Ala Glu Val Glu Lys Pro Gly Ala Ser Val
20 25 30
Lys Ile Ser Cys Lys Ala Ser Gly Ser Ser Phe Thr Gly Tyr Asn Met
35 40 45
Asn Trp Val Arg Gln Asn Ile Gly Lys Ser Leu Glu Trp Ile Gly Ala
50 55 60
Ile Asp Pro Tyr Tyr Gly Gly Thr Ser Tyr Asn Gln Lys Phe Lys Gly
65 70 75 80
Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr Met His
85 90 95
Leu Lys Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Val Ser
100 105 110
Gly Met Glu Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly
115 120 125
Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Ser Glu Gly Ser Thr Lys
130 135 140
Gly Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro
145 150 155 160
Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His
165 170 175
Arg Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln
180 185 190
Ser Pro Lys Leu Leu Ile His Lys Val Ser Asn Arg Phe Ser Gly Val
195 200 205
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys
210 215 220
Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln
225 230 235 240
Ser Thr His Val Pro Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu
245 250 255
Leu Lys Ala Ala Ala Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp
260 265 270
Asn Glu Lys Ser Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu
275 280 285
Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu
290 295 300
Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val
305 310 315 320
Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His
325 330 335
Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys
340 345 350
His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser
355 360 365
Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Val Val Lys Arg
370 375 380
Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro
385 390 395 400
Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu
405 410 415
Glu Glu Glu Gly Gly Cys Glu Leu Gly Gly Gly Gly Ser Gly Gly Gly
420 425 430
Gly Ser Gly Gly Gly Gly Ser Arg Val Lys Phe Ser Arg Ser Ala Asp
435 440 445
Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn
450 455 460
Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg
465 470 475 480
Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly
485 490 495
Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu
500 505 510
Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu
515 520 525
Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His
530 535 540
Met Gln Ala Leu Pro Pro Arg Thr Ser Gly Ser Gly Ala Thr Asn Phe
545 550 555 560
Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met
565 570 575
Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro
580 585 590
Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu Asp
595 600 605
Gly Lys Lys Val Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe
610 615 620
Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala
625 630 635 640
Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr
645 650 655
Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala Thr
660 665 670
Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Gly Gly Gly Gly Ser
675 680 685
Gly Gly Gly Gly Ser Gly Ala Met Val Gly Ala Leu Glu Ser Leu Arg
690 695 700
Gly Asn Ala Asp Leu Ala Tyr Ile Leu Ser Met Glu Pro Cys Gly His
705 710 715 720
Cys Leu Ile Ile Asn Asn Val Asn Phe Cys Arg Glu Ser Gly Leu Arg
725 730 735
Thr Arg Thr Gly Ser Asn Ile Asp Cys Glu Lys Leu Arg Arg Arg Phe
740 745 750
Ser Ser Leu His Phe Met Val Glu Val Lys Gly Asp Leu Thr Ala Lys
755 760 765
Lys Met Val Leu Ala Leu Leu Glu Leu Ala Arg Gln Asp His Gly Ala
770 775 780
Leu Asp Cys Cys Val Val Val Ile Leu Ser His Gly Cys Gln Ala Ser
785 790 795 800
His Leu Gln Phe Pro Gly Ala Val Tyr Gly Thr Asp Gly Cys Pro Val
805 810 815
Ser Val Glu Lys Ile Val Asn Ile Phe Asn Gly Thr Ser Cys Pro Ser
820 825 830
Leu Gly Gly Lys Pro Lys Leu Phe Phe Ile Gln Ala Cys Gly Gly Glu
835 840 845
Gln Lys Asp His Gly Phe Glu Val Ala Ser Thr Ser Pro Glu Asp Glu
850 855 860
Ser Pro Gly Ser Asn Pro Glu Pro Asp Ala Thr Pro Phe Gln Glu Gly
865 870 875 880
Leu Arg Thr Phe Asp Gln Leu Asp Ala Ile Ser Ser Leu Pro Thr Pro
885 890 895
Ser Asp Ile Phe Val Ser Tyr Ser Thr Phe Pro Gly Phe Val Ser Trp
900 905 910
Arg Asp Pro Lys Ser Gly Ser Trp Tyr Val Glu Thr Leu Asp Asp Ile
915 920 925
Phe Glu Gln Trp Ala His Ser Glu Asp Leu Gln Ser Leu Leu Leu Arg
930 935 940
Val Ala Asn Ala Val Ser Val Lys Gly Ile Tyr Lys Gln Met Pro Gly
945 950 955 960
Cys Phe Asn Phe Leu Arg Lys Lys Leu Phe Phe Lys Thr Ser Ala Ser
965 970 975
<210> 14
<211> 987
<212> PRT
<213> 人工合成
<400> 14
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro Ala Phe
1 5 10 15
Leu Leu Ile Pro Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val
20 25 30
Leu Pro Gly Asp Ser Leu Arg Leu Ser Cys Ala Thr Ser Glu Phe Thr
35 40 45
Phe Thr Asp Tyr Tyr Met Thr Trp Val Arg Gln Pro Pro Arg Lys Ala
50 55 60
Leu Glu Trp Leu Gly Phe Ile Arg Asn Arg Ala Asn Gly Tyr Thr Thr
65 70 75 80
Glu Tyr Asn Pro Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
85 90 95
Ser Gln Ser Ile Leu Tyr Leu Gln Met Asn Thr Leu Arg Thr Glu Asp
100 105 110
Ser Ala Thr Tyr Tyr Cys Ala Arg Val Ser Asn Trp Ala Phe Asp Tyr
115 120 125
Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Ser Thr Ser Gly
130 135 140
Ser Gly Lys Pro Gly Ser Ser Glu Gly Ser Thr Lys Gly Asp Val Val
145 150 155 160
Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly Asp Gln Ala
165 170 175
Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Lys Asn Asn Gly Asn
180 185 190
Thr Phe Leu His Trp Tyr Leu Gln Lys Ser Gly Gln Ser Pro Lys Leu
195 200 205
Leu Ile Tyr Lys Val Ser Asn Arg Leu Ser Gly Val Pro Asp Arg Phe
210 215 220
Ser Gly Ser Gly Ser Gly Thr Tyr Phe Thr Leu Lys Ile Ser Arg Val
225 230 235 240
Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gln Ser Thr His Ile
245 250 255
Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Ala Ala Ala
260 265 270
Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn
275 280 285
Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu
290 295 300
Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly
305 310 315 320
Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe
325 330 335
Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn
340 345 350
Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr
355 360 365
Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Ala Ser Gly Gly Gly
370 375 380
Gly Ser Gly Gly Gly Gly Ser Val Val Lys Arg Gly Arg Lys Lys Leu
385 390 395 400
Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln
405 410 415
Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly
420 425 430
Cys Glu Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
435 440 445
Gly Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln
450 455 460
Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu
465 470 475 480
Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly
485 490 495
Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu
500 505 510
Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly
515 520 525
Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser
530 535 540
Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro
545 550 555 560
Pro Arg Thr Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln
565 570 575
Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Gly Val Gln Val Glu
580 585 590
Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr
595 600 605
Cys Val Val His Tyr Thr Gly Met Leu Glu Asp Gly Lys Lys Val Asp
610 615 620
Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys Phe Met Leu Gly Lys Gln
625 630 635 640
Glu Val Ile Arg Gly Trp Glu Glu Gly Val Ala Gln Met Ser Val Gly
645 650 655
Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr
660 665 670
Gly His Pro Gly Ile Ile Pro Pro His Ala Thr Leu Val Phe Asp Val
675 680 685
Glu Leu Leu Lys Leu Glu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
690 695 700
Gly Ala Met Val Gly Ala Leu Glu Ser Leu Arg Gly Asn Ala Asp Leu
705 710 715 720
Ala Tyr Ile Leu Ser Met Glu Pro Cys Gly His Cys Leu Ile Ile Asn
725 730 735
Asn Val Asn Phe Cys Arg Glu Ser Gly Leu Arg Thr Arg Thr Gly Ser
740 745 750
Asn Ile Asp Cys Glu Lys Leu Arg Arg Arg Phe Ser Ser Leu His Phe
755 760 765
Met Val Glu Val Lys Gly Asp Leu Thr Ala Lys Lys Met Val Leu Ala
770 775 780
Leu Leu Glu Leu Ala Arg Gln Asp His Gly Ala Leu Asp Cys Cys Val
785 790 795 800
Val Val Ile Leu Ser His Gly Cys Gln Ala Ser His Leu Gln Phe Pro
805 810 815
Gly Ala Val Tyr Gly Thr Asp Gly Cys Pro Val Ser Val Glu Lys Ile
820 825 830
Val Asn Ile Phe Asn Gly Thr Ser Cys Pro Ser Leu Gly Gly Lys Pro
835 840 845
Lys Leu Phe Phe Ile Gln Ala Cys Gly Gly Glu Gln Lys Asp His Gly
850 855 860
Phe Glu Val Ala Ser Thr Ser Pro Glu Asp Glu Ser Pro Gly Ser Asn
865 870 875 880
Pro Glu Pro Asp Ala Thr Pro Phe Gln Glu Gly Leu Arg Thr Phe Asp
885 890 895
Gln Leu Asp Ala Ile Ser Ser Leu Pro Thr Pro Ser Asp Ile Phe Val
900 905 910
Ser Tyr Ser Thr Phe Pro Gly Phe Val Ser Trp Arg Asp Pro Lys Ser
915 920 925
Gly Ser Trp Tyr Val Glu Thr Leu Asp Asp Ile Phe Glu Gln Trp Ala
930 935 940
His Ser Glu Asp Leu Gln Ser Leu Leu Leu Arg Val Ala Asn Ala Val
945 950 955 960
Ser Val Lys Gly Ile Tyr Lys Gln Met Pro Gly Cys Phe Asn Phe Leu
965 970 975
Arg Lys Lys Leu Phe Phe Lys Thr Ser Ala Ser
980 985

Claims (10)

1.一种基于GD2嵌合抗原受体，其特征在于，所述嵌合抗原受体包括抗原结合结构域、跨膜结构域、共刺激信号传导结构域、CD3ζ信号传导结构域和自毁结构域串联而成；
其中，所述抗原结合结构域结合肿瘤表面抗原，所述肿瘤表面抗原为GD2，所述抗原结合结构域为针对肿瘤表面抗原GD2的单链抗体，所述肿瘤表面抗原GD2的单链抗体的氨基酸序列选自：
(a)如SEQ ID NO.1-3所示的氨基酸序列；或者，
(b)如SEQ ID NO.1-3所示的氨基酸序列经过一个或多个氨基酸的取代、添加或缺失而形成的，能够特异结合在嵌合抗原受体上，具有结合GD2和诱导T细胞信号传导功能的氨基酸序列。
2.根据权利要求1所述的嵌合抗原受体，其特征在于，所述如SEQ ID NO.1-3所示的氨基酸序列经过一个或多个氨基酸的取代、添加或缺失而形成的氨基酸序列与SEQ ID NO.1-3所示的氨基酸序列具有至少90％同一性，优选为95％同一性的氨基酸序列。
3.根据权利要求1或2所述的嵌合抗原受体，其特征在于，所述跨膜结构域为CD28跨膜结构域和/或CD8α跨膜结构域；
优选地，所述共刺激信号传导结构域为CD28信号传导结构域和4-1BB信号传导结构域的组合；
优选地，所述共刺激信号传导结构域的氨基酸序列如SEQ ID NO.4所示。
4.根据权利要求1-3中任一项所述的嵌合抗原受体，其特征在于，，所述自毁结构域为包含胱天蛋白酶9结构域；
优选地，所述胱天蛋白酶9结构域的氨基酸序列如SEQ ID NO.9所示；
优选地，所述自毁结构域通过2A序列与CD3ζ信号传导结构域相串联。
5.根据权利要求1-4中任一项所述的嵌合抗原受体，其特征在于，所述嵌合抗原受体包括信号肽、抗原结合结构域、跨膜结构域、共刺激信号传导结构域、CD3ζ信号传导结构域、2A序列和自毁结构域串联而成；
优选地，所述嵌合抗原受体为Secretory信号肽，GD2抗原结合结构域，CD8α和/或CD28跨膜结构域，CD28信号传导结构域、4-1BB信号传导结构域，CD3ζ信号传导结构域、2A序列和胱天蛋白酶9结构域串联而成；
优选地，所述嵌合抗原受体为Secretory-GD2-CD28-4-1BB-CD3ζ-2A-FBKP.Casp9；
优选地，所述嵌合抗原受体Secretory-GD2-CD28-4-1BB-CD3ζ-2A-FBKP.Casp9的氨基酸序列如SEQ ID NO.12-14所示。
6.一种病毒载体，其特征在于，包括权利要求1-5中任一项所述的嵌合抗原受体；
优选地，所述病毒载体为慢病毒载体和/或逆转录病毒载体，优选为慢病毒载体。
7.一种T细胞，其特征在于，采用如权利要求1-5中任一项所述的嵌合抗原受体通过其编码的核酸序列转染到T细胞中表达；
优选地，所述转染的方式为通过病毒载体和/或真核表达质粒转染到T细胞，优选为通过病毒载体转染到T细胞。
8.一种重组慢病毒，其特征在于，将包含如权利要求6所述的病毒载体与包装辅助质粒pNHP和pHEF-VSVG共转染哺乳细胞得到的重组慢病毒；
优选地，所述哺乳细胞为293细胞、293T细胞或TE671细胞中的任意一种或至少两种的组合。
9.一种组合物，其特征在于，所述组合物包括如权利要求1-7中任一项所述的嵌合抗原受体和/或如权利要求8所述的重组慢病毒。
10.如权利要求1-7中任一项所述的嵌合抗原受体、如权利要求8所述的重组慢病毒或如权利要求9所述的组合物在制备嵌合抗原受体T细胞或免疫细胞及其在肿瘤治疗药物中的应用；
优选地，所述肿瘤为GD2特异抗原表达的肿瘤疾病；
优选地，所述GD2特异抗原表达的肿瘤疾病为神经母细胞瘤。
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CN116769723A (zh) * 2023-08-09 2023-09-19 山东省成体细胞产业技术研究院有限公司 一种gd2嵌合抗原受体修饰的t细胞及其应用

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